CN103710380B - The application in promoting plant vitamin C synthesis of the OsVTC1-3 albumen - Google Patents
The application in promoting plant vitamin C synthesis of the OsVTC1-3 albumen Download PDFInfo
- Publication number
- CN103710380B CN103710380B CN201310699609.8A CN201310699609A CN103710380B CN 103710380 B CN103710380 B CN 103710380B CN 201310699609 A CN201310699609 A CN 201310699609A CN 103710380 B CN103710380 B CN 103710380B
- Authority
- CN
- China
- Prior art keywords
- carrier
- osvtc1
- plant
- albumen
- obtains
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses the application in promoting plant vitamin C synthesis of a kind of OsVTC1 3 albumen.A kind of interference carrier disclosed by the invention, this carrier is between Xho I and Bgl II site that the DNA fragmentation shown in SEQ ID No.3 inserts the carrier pUCCRNAi that sets out, and obtains intermediate carrier 1；The reverse complementary sequence of the DNA fragmentation shown in SEQ ID No.3 is inserted between Sal I and BamH I site of intermediate carrier 1, obtain intermediate carrier 2；With Pst I enzyme action intermediate carrier 2, obtain DNA fragmentation；Pst I enzyme action pCAMBIA2300, obtains carrier large fragment；DNA fragmentation is connected with carrier large fragment, finally gives interference carrier.Present invention demonstrates that OsVTC1 3 albumen has important function in plant Vc synthesizes.
Description
Technical field
The present invention relates to the application in promoting plant vitamin C synthesis of a kind of OsVTC1-3 albumen.
Background technology
Vitamin C (Vitamin C, Vc) is important antioxidant and coenzyme in animal and plant body, but people cannot close
Become Vc, need to absorb from diet.Plant is the important sources of needed by human body Vc but plant particularly grain is made
In thing, Vc content is the lowest, so by Vc content in the approach raising plant of biotechnology to improving nutritive value of crops
There is important function.
In animal, Vc synthesis path is clearer.Its building-up process is through UDPG, UDP-D-by D-Glucose
Glucuronic acid, D-Glucose aldehydic acid, L-GuA, L-gulose-Isosorbide-5-Nitrae-lactone, finally at gulose lactone
Vc is synthesized under oxidasic effect.The mankind and some animals become different owing to encoding the oxidasic gene of gulose lactone
And Vc can not be synthesized.Different from animal, in plant, Vc route of synthesis is more complicated.Research shows may deposit in plant
Close at a plurality of Vc such as glucuronate pathway, osone approach, inositol pathway, gulose approach and L-galactose approach
One-tenth approach.Although different approaches is proved in different experiments, but the experiment in model plant arabidopsis shows half
Lactose approach is the main path of plant Vc synthesis.
Galactose approach synthesis Vc relates to mannose-6-phosphate isomerase, Phosphomannomutase, GDP-mannose
Pyrophosphorylase, GDP-mannose-3', 5'-epimerase, GDP-L-galactose phosphorylase, galactose-1-phosphate
The multiple enzyme of enzyme, galactose dehydrogenase and galactanolactone dehydrogenase.In arabidopsis, galactose approach is with phosphofructose
For substrate, under the catalysis of relevant enzyme, through phosphomannose, GDP-mannose, GDP-galactose, phosphoric acid galactose,
Then galactose lactone, then generated Vc by L-1,4-galactose lactone under the effect of galactanolactone dehydrogenase.
Having multiple key regulatory site in this approach, wherein catalysis 1-phosphomannose generates the GDP-manna of GDP-mannose
Sugar pyrophosphorylase VTC1 plays important regulating and controlling effect in this route of synthesis.The Point mutont vtc1-1 of VTC1 because
The GDP-mannose pyrophosphorylase of VTC1 reduces and suppresses arabidopsis Vc to synthesize, and its Vc content only has wild type
25-30%；And VTC1 afunction mutant, then cannot complete life cycle because of Vc synthesis suppression.So VTC1 coding
GDP-mannose pyrophosphorylase plant Vc synthesize in there is important function.
At present, although Vc route of synthesis is clearer in model plant arabidopsis, but in cereal crops are such as Oryza sativa L.
Vc route of synthesis and regulatory site are not clear.
Summary of the invention
It is an object of the invention to provide the application in promoting plant vitamin C synthesis of a kind of OsVTC1-3 albumen.
A kind of interference carrier that the present invention provides, this carrier is to be inserted by the DNA fragmentation forward shown in SEQ ID No.3
Set out between Xho I and Bgl II site of carrier pUCCRNAi, obtain intermediate carrier 1；Shown in SEQ ID No.3
DNA fragmentation reverse complementary sequence forward insert intermediate carrier 1 Sal I and BamH I site between, obtain centre
Carrier 2；With Pst I enzyme action intermediate carrier 2, obtain DNA fragmentation；Pst I enzyme action pCAMBIA2300, obtains carrier
Large fragment；DNA fragmentation is connected with carrier large fragment, finally gives interference carrier.
A kind of method preparing the transgenic plant that ascorbic acid biosynthesis ability improves falls within protection scope of the present invention,
Comprise the steps:
Being imported by the encoding gene of albumen shown in SEQ ID No.2 sets out in plant, obtains transgenic plant；With set out
Plant is compared, and the ascorbic acid biosynthesis ability of transgenic plant improves.
In said method, described encoding gene imports in described plant by recombinant expression carrier, described restructuring table
Reaching carrier is that the multiple clone site that described encoding gene inserts the carrier pCAMBIA1307 that sets out obtains.
In any of the above-described described method, the nucleotide sequence of described encoding gene is as shown in SEQ ID No.1；
Described plant is specially arabidopsis.
A kind of method preparing the transgenic plant that ascorbic acid biosynthesis ability reduces falls within protection scope of the present invention,
Comprise the steps:
Being imported by interference carrier sets out in plant, obtains transgenic plant；Compared with the plant that sets out, transgenic plant
Ascorbic acid biosynthesis ability reduces；
Described interference carrier is above-mentioned interference carrier；
Described plant is specially Oryza sativa L..
The application in preparing the transgenic plant that ascorbic acid biosynthesis ability improves of the following arbitrary material falls within the present invention
Protection domain:
(1) albumen shown in SEQ ID No.2；
(2) encoding gene of albumen shown in SEQ ID No.2；
(3) recombinant vector, expression cassette, transgenic cell line or the recombinant bacterium containing (2) described encoding gene.
In above-mentioned application, the nucleotide sequence of described encoding gene is as shown in SEQ ID No.1.
In any of the above-described described application, described plant is arabidopsis.
Albumen shown in SEQ ID No.2 falls within the protection of the present invention as the application of GDP-mannose pyrophosphorylase
Scope.
OsVTC1-3 albumen can be catalyzed mannose-1-phosphate and generate GDP-mannose in vitro, shows OsVTC1-3
Albumen has GDP-mannose pyrophosphorylase activity, and it may participate in galactose approach and regulate and control Vc synthesis in vivo.
OsVTC1-3 can improve Vc content in the single-point mutants vtc1-1 of arabidopsis VTC1.It addition, in interference Oryza sativa L.
The expression of OsVTC1-3 significantly suppress the synthesis of Oryza sativa L. Vc.Visible OsVTC1-3 albumen has in plant Vc synthesizes
Play an important role.
Accompanying drawing explanation
Fig. 1 be OsVTC1-3 prokaryotic expression carrier pET-30a (+)-OsVTC1-3 builds schematic diagram.
Fig. 2 is purification and the GDP-mannose pyrophosphorylase activity of OsVTC1-3 albumen of OsVTC1-3 albumen
Detection.
Fig. 3 is that OsVTC1-3 gene overexpression carrier pCAMBIA1307-OsVTC1-3 builds schematic diagram.
Fig. 4 is the Western blot detection of OsVTC1-3 gene overexpression plant.
Fig. 5 is that OsVTC1-3 gene interference expression vector pCAMBIA2300-RNAi-OsVTC1-3 builds schematic diagram.
Fig. 6 is OsVTC1-3 gene expression testing result in OsVTC1-3 gene interference plant.
Fig. 7 is OsVTC1-3 gene overexpression plant and the testing result of Vitamin C content in interference plant.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Flower 17(Zhonghua17 in Oryza sativa L.) at document " Liu D, Chen X, Liu J, Ye J, Guo Z. (2012)
The rice ERF transcription factor OsERF922negatively regulates resistance to
Magnaporthe oryzae and salt tolerance.J Exp Bot.63:3899-3911. " disclosed in mistake,
The public can obtain from Biological Technology institute, Chinese Academy of Agricultural Sciences.
The single-point mutants vtc1-1 of arabidopsis ascorbic acid biosynthesis key gene VTC1 document " Conklin PL,
Saracco SA,Norris SR,Last RL.(2000)Identification of ascorbic
Acid-deficient Arabidopsis thaliana mutants.Genetics.154:847-856. " disclosed in
Crossing, the public can obtain from Biological Technology institute, Chinese Academy of Agricultural Sciences.
BCA protein quantification test kit is purchased from Beijing CoWin Bioscience Co., Ltd., and catalog number is CW0014.
PET-30a (+) purchased from Beijing Bichenglan Biotechnology Co., Ltd., catalog number is S18-12.
1-P-mannose(mannose-1-phosphate) purchased from Sigma company, catalog number is P6556.
20mM TEAA buffer believes international corporation purchased from Jiade, and catalog number is SP5890.
PCAMBIA1307 is purchased from Ji Ran bio tech ltd, Shanghai, and catalog number is JR13080311.
Agrobacterium LBA4404 is purchased from Hang Zhi bio tech ltd, and catalog number is AABV02-03.
Inducing culture (NB culture medium) is purchased from Ke Min bio tech ltd, Shanghai, and article No. is BS1309.
Subculture medium is to add 2mg/L2,4-D in NB culture medium to make.
100uM AS+AA fluid medium is added 2mg/L2,4-D and 100uM/L AS by NB culture medium and makes.
PUCCRNAi is at document " Gan D, Zhang J, Jiang H, Jiang T, Zhu S, Cheng B (2010)
Bacterially expressed dsRNA protects maize against SCMV infection.Plant Cell
Rep.29:1261-1268. " mistake disclosed in, the public can obtain from Biological Technology institute, Chinese Academy of Agricultural Sciences.
PCAMBIA2300 is purchased from Beijing prosperity Bioisystech Co., Ltd of ancient cooking vessel state, and catalog number is MCV036.
Embodiment 1, the expression in escherichia coli of the OsVTC1-3 albumen
One, the structure of OsVTC1-3 prokaryotic expression carrier
(1) genomic DNA spending 17 is extracted in Oryza sativa L..
(2) following primer is designed and synthesized:
Forward primer: 5'-GGCATATGACCATGAAGGCGCTCA-3'
Downstream primer: 5'-ATGTCGACCATGACAATCTCGGGCTT-3'
(sequence shown in underscore is enzyme action recognition site)
(3) with the genomic DNA of step () as template, with forward primer and the downstream primer of step (two)
Carrying out PCR amplification for primer, PCR primer is OsVTC1-3 gene, the sequence of this gene such as SEQ ID No.1 institute
Showing, the sequence of OsVTC1-3 albumen is as shown in SEQ ID No.2.
(4) PCR primer that NdeI Yu SalI double digestion step (three) obtains, obtains genetic fragment；NdeI with
SalI double digestion pET-30a (+), obtain carrier large fragment；Genetic fragment is connected with carrier large fragment, is recombinated
Plasmid, by its named pET-30a (+)-OsVTC1-3, sequencing result is correct, and it builds schematic diagram as shown in Figure 1.
Two, the purification of OsVTC1-3 albumen
By pET-30a (+)-OsVTC1-3 electricity convert e. coli bl21, the positive colony of acquisition is inoculated in LB cultivate
Base, adds the expression of the IPTG induction OsVTC1-3 albumen of final concentration of 0.5 μM in the medium,
28 DEG C, 200rpm cultivates 1 hour, collects bacterium solution, liquid nitrogen grinding smudge cells, obtains protein crude extract；Simultaneously by being not added with
The culture medium of IPTG obtains reference protein crude extract by same method；By protein crude extract Bio-Rad albumen low pressure
Tomographic system purification, obtains purified components.
Protein crude extract, reference protein crude extract and purified components are carried out PAGE gel electrophoresis result such as Fig. 2 A
Shown in.
In Fig. 2 A, marker is albumen Marker, and-IPTG represents reference protein crude extract；It is thick that+IPTG represents albumen
Extract；Purified represents purified components (i.e. OsVTC1-3 albumen).
Fig. 2 A shows, the molecular weight of OsVTC1-3 albumen is 36.2kD.
By pET-30a (+) plasmid carries out the experiment of step 2, occur without purpose band.
Three, OsVTC1-3 Protein G DP-mannose pyrophosphorylase Activity determination
GDP-mannose pyrophosphorylase can be catalyzed mannose-1-phosphate and generate GDP-mannose.
The OsVTC1-3 protein sample (one) step 2 purification obtained and pET-30a (+) the His label egg expressed
(reference protein) is separately added in bag filter in vain, desalts with the dialysis of 100mM Tris-HCl dialysis buffer liquid.Dialysis
The displacement of every 1 hour of buffer once, replace 5 times, each albumen subpackage after dialysing be stored in-80 DEG C stand-by.
(2) by the OsVTC1-3 albumen obtained and reference protein BCA protein quantification kit measurement protein concentration.
(3) take each albumen that 1 μ g purification obtains respectively to carry out GDP-mannose pyrophosphorylase by following system and live
Property detection.
Enzyme reaction system alive is as follows:
Adding water to cumulative volume is 50 μ l.
37 DEG C of reaction 1h.Boiling water bath 2min terminates reaction.
(4) synthetic quantity of HPLC detection GDP-mannose
Chromatographic column used be Waters company C-18 chromatographic column (model is wat046980, and filler is high-purity silica gel,
Chromatographic column specification is 250 × 4.6mm)
Elution buffer is as follows:
Elution buffer I:20mM TEAA buffer and acetonitrile mix according to volume ratio 2:98.
Elution buffer II:20mM TEAA buffer and acetonitrile mix according to volume ratio 5:95.
Elution buffer III:20mM TEAA buffer and acetonitrile mix according to volume ratio 10:90.
Elution requirement is: elution buffer I linear elution 12min, then with elution buffer II linear elution 5min,
Finally with elution buffer III linear elution 5min.
GDP-mannose standard sample is made 10mM mother solution, then mother solution is pressed
The gradient dilution of 0,100,500,1000,1500,2000,2500,3000nM becomes the GDP-mannose standard of variable concentrations
Sample, respectively takes 25 μ l standard samples, and column temperature 25 DEG C, flow velocity 1ml/min crosses chromatographic column, then by above-mentioned eluting bar
Part carries out eluting, and at the appearance time of 254nm examination criteria sample with calculate peak area, with the time as abscissa (point
Clock), GDP-mannose concentration is that vertical coordinate is mapped (nM), draws GDP-mannose standard curve.
Each reactant liquor that step (three) obtains is taken 25 μ l volume loadings, column temperature 25 DEG C, flow velocity 1ml/min, 254nm
The peak area of GDP-mannose in detection reactant liquor.
The GDP-mannose content (nM) in reactant liquor is calculated according to GDP-mannose standard curve.
GDP-mannose pyrophosphorylase activity (nM/hr/mg the albumen)=GDP-mannose of OsVTC1-3 albumen contains
OsVTC1-3 protein content (mg) in amount 1 hour (nM)/enzyme reaction time (hr)/sample.
Result as shown in Figure 2 B, live as 1478.2+52.7 by the GDP-mannose pyrophosphorylase of OsVTC1-3 albumen
(nM/hr/mg albumen).
GDP-mannose pyrophosphorylase activity (nM/hr/mg albumen)=GDP-mannose content (nM) of reference protein
His label protein amount (mg) in 1 hour/enzyme reaction time (hr)/sample.
PET-30a (+) the GDP-mannose pyrophosphorylase of His label protein (reference protein) expressed lives and be only 3.7+
1.2 (nM/hr/mg albumen).
To sum up show that OsVTC1-3 has GDP-mannose pyrophosphorylase activity.
Embodiment 2, OsVTC1-3 gene overexpression vector arabidopsis
One, the structure of over-express vector
(1) genomic DNA spending 17 is extracted in Oryza sativa L..
(2) following primer is designed and synthesized:
Forward primer: 5'-GCTCTAGAACCATGAAGGCGCTCATT-3'
Downstream primer: 5'-ATGTCGACCATGACAATCTCGGGCTT-3'
(sequence shown in underscore is enzyme action recognition site)
(3) with the genomic DNA of step () as template, draw with the forward primer in step (two) and downstream
Thing is that primer carries out PCR amplification, obtains OsVTC1-3 gene.
(4) PCR primer obtained with Xba I and Sal I double digestion step (three), obtains genetic fragment；Xba
I Yu Sal I double digestion pCAMBIA1307, obtains carrier large fragment；Genetic fragment is connected with carrier large fragment,
To recombiant plasmid, by its named pCAMBIA1307-OsVTC1-3, this plasmid sending sequencing result correct, it builds
Schematic diagram is as shown in Figure 3.
Two, pCAMBIA1307-OsVTC1-3 is imported in Agrobacterium LBA4404 by electricity method for transformation, recombinated
Agrobacterium, by its named LBA4404/pCAMBIA1307-OsVTC1-3.
Three, over-express vector arabidopsis thaliana transformation
(1) the arabidopsis ascorbic acid biosynthesis key gene VTC1 that upgrowth situation is good, have more petal is taken
Single-point mutants vtc1-1, convert the previous day water.
(2) by recombinational agrobacterium LBA4404/pCAMBIA1307-OsVTC1-3 in 28 DEG C of overnight incubation, extremely
OD600=0.8,6000rpm are centrifuged 6min, collect thalline.
(3) bacterial sediment is suspended in freshly prepared conversion buffer, to final concentration 0D600=0.4, make outstanding
Supernatant liquid.
(4) cut off the flower bloomed or existing fruit pod when converting, topple over little basin, it is ensured that arabidopsis is whole
Petal is all submerged in suspension prepared by step (three), soaks about 40s.
(5) suck the liquid that surrounding plants is unnecessary, plant is lain against one seal capsule in keep humidity,
Lucifuge is overnight.
Within (six) second days, plant is taken out, vertically, transfers to 20 DEG C, grow under 16h illumination/8h dark condition,
Obtain T1 for seed.
(7) T1 is laid in the screening culture medium containing 40ug/mL hygromycin for seed, 4 DEG C of vernalization 48h-72h,
Move on to phjytotron, grow two weeks under 16h illumination/8h dark condition.
(8) wait to grow green transgenic resistance seedling (transgenic plant be green seedling and root longer；Non-turn base
Because plant is essentially etiolated seedling or green unrooted seedling) transplant continued growth of burying afterwards, it is thus achieved that and T1 is for transfer-gen plant.
(9) screening T1 has the transfer-gen plant of 3:1 segregation ratio for hygromycin resistance and the non-resistance seed of Seedling
And adding generation, obtain T3 for transgenic arabidopsis.
To wildtype Arabidopsis thaliana, the single-point mutants vtc1-1 of arabidopsis ascorbic acid biosynthesis key gene VTC1 and
Three strain T3 carry out Western blot for transgenic arabidopsis (OE-1, OE-2 and OE-3), detect OsVTC1-3
The expression of albumen (tape label), result is as shown in Figure 4.
In Fig. 4, WT is wildtype Arabidopsis thaliana；Vtc1-1 is the list of arabidopsis ascorbic acid biosynthesis key gene VTC1
Point mutont vtc1-1；OE-1, OE-2 and OE-3 are three strain T3 generations to turn OsVTC1-3 over-express vector
The arabidopsis of pCAMBIA1307-OsVTC1-3.
Fig. 4 shows, nothing in the single-point mutants of wildtype Arabidopsis thaliana and arabidopsis ascorbic acid biosynthesis key gene VTC1
The expression of OsVTC1-3 albumen, three strain T3 are for turning OsVTC1-3 over-express vector pCAMBIA1307-OsVTC1-3's
Arabidopsis (OE-1, OE-2 and OE-3) has the expression of OsVTC1-3 albumen.
T3 is applied to the functional analysis in plant of the OsVTC1-3 albumen for transgenic arabidopsis.
Embodiment 3, OsVTC1-3 gene interference carrier rice transformation
One, the structure of interference carrier
(1) genomic DNA spending 17 is extracted in Oryza sativa L..
(2) following primer is designed and synthesized:
Forward primer: 5'-AACTCGAGGAGGCGAGGCGAAGGATT-3'；
Downstream primer: 5'-GGAGATCTCAACCATATAGCCTGATGACA-3'。
(sequence shown in underscore is enzyme action recognition site)
(3) with the genomic DNA of step () as template, with forward primer and the downstream primer of step (two)
Carry out PCR amplification for primer, obtain pcr amplification product, the nucleotide sequence of this DNA molecular such as SEQ ID No.3
Shown in, this fragment does not comprise OsVTC1-3 gene cDNA fragment, is positioned at 3 ' UTR districts of OsVTC1-3 gene.
(4) PCR primer that Xho I and Bgl II double digestion step (three) obtains, obtains OsVTC1-3 gene
3 ' UTR fragments；Xho I and Bgl II double digestion pUCCRNAi, obtains carrier large fragment 1；By OsVTC1-3 gene
3 ' UTR fragments be connected with carrier large fragment 1, obtain intermediate carrier 1.
Sal I and BamH I double digestion intermediate carrier 1, obtain carrier large fragment 2；Xho I and Bgl II double digestion step
(3) PCR primer obtained, obtains 3 ' UTR fragments of OsVTC1-3 gene；By carrier large fragment 2 and OsVTC1-3
3 ' UTR fragments of gene connect, and obtaining intermediate carrier 2(Xho I with BamH I, Bgl II and Sal I is isocaudarner).
Pst I enzyme action intermediate carrier 2, obtains 3 ' UTR fragments of the OsVTC1-3 gene of two-way complementation；Pst I enzyme
Cut pCAMBIA2300, obtain carrier large fragment 3；By 3 ' UTR fragment and loads of the OsVTC1-3 gene of two-way complementation
Body large fragment 3 connects, and obtains recombiant plasmid, and by its named pCAMBIA2300-RNAi-OsVTC1-3, it builds
Schematic diagram is as shown in Figure 5.
Two, pCAMBIA2300-RNAi-OsVTC1-3 is imported in Agrobacterium LBA4404 with electricity method for transformation, obtain
Recombinational agrobacterium, by its named LBA4404/pCAMBIA2300-RNAi-OsVTC1-3.
Three, interference expression vector rice transformation
(1) by with the Oryza sativa L. after volumn concentration 75% ethanol sterilizing is spent the seed of 17 on inducing culture,
28 DEG C of dark culturing.After two weeks, callus is proceeded to new inducing culture, 28 DEG C of subculture light culture, altogether subculture
Light culture 2 generation.
(2) the embryo callus subculture granule naturally disperseed in third generation subcultured callus, color is yellowish is placed in subculture training
Supporting on base, 28 DEG C of light culture 3 days, the callus obtained is for the conversion of recombinational agrobacterium.
(3) recombinational agrobacterium LBA4404/pCAMBIA2300-RNAi-OsVTC1-3 is suspended in 100uM AS+AA
(OD in fluid medium600It is 0.3), then the callus that step (two) obtains is placed in bacterium suspension immersion
10-20 minute, it is shaken gently for therebetween, in 28 DEG C of overnight incubation, to OD600=0.8,6000rpm are centrifuged 6min, receive
Collection thalline.Bacterial sediment is suspended in freshly prepared conversion buffer, to final concentration 0D600=0.3-0.6。
(4) outwelling bacterium solution, the careful callus filter paper that takes out blots unnecessary bacterium solution, is transferred to by callus altogether
In culture medium, 21 DEG C of-22 DEG C of light culture 3 days.
(5) selecting the callus after co-culturing, filter paper is laid in screening culture medium after being filtered dry surface steam and (contains
50mg/L hygromycin) on, 28 DEG C carry out light culture.When 2 weeks, subculture is once, altogether subculture 2 times.
(6) growth step (five) obtained is vigorous, is creamy white or yellowish fresh callus goes to pre-
Division culture medium, 28 DEG C of light culture about 1 week, then 28 DEG C of illumination cultivation, about two weeks subcultures once, are divided
Change Seedling.
(7) seedling differentiation that growing way is good is moved on root media (bottled).Cultivate intermediate house after 1-2 week,
Obtain T0 for transfer-gen plant and the seed of T0 transfer-gen plant.
Use hygromycin selection plates to sprout in the seed of T0 transfer-gen plant, confirm that hygromycin resistance has 3:1 and divides
From than strain, then T1 transfer-gen plant therefrom is gone to soil incubation until ripe.Base is turned by obtain
Because of material adding generation, obtain T3 for transgenic paddy rice.
Extract in wild rice and spend 17 and T3 for the RNA of transgenic paddy rice, carry out Q-PCR experiment, detection
The relative expression quantity of OsVTC1-3 gene, result is as shown in Figure 6.
In Fig. 6, WT is to spend 17 in wild rice；RI-2 and RI-4 is two strain T3 generations to turn OsVTC1-3 interference to carry
The Oryza sativa L. of body pCAMBIA2300-RNAi-OsVTC1-3.
Fig. 6 shows, and spends compared with in the of 17 in wild rice, RI-2 and RI-4 this two strain T3 is for transgenic paddy rice
The relative expression quantity of OsVTC1-3 gene is remarkably decreased.
The T3 obtained is utilized to analyze OsVTC1-3 albumen function in plant for transgenic paddy rice.
Vitamin C content detection in embodiment 4, transfer-gen plant
One, choose 3 week old T3 for transgenic paddy rice and wild rice are spent 17 root and T3 for transgenic arabidopsis,
Wildtype Arabidopsis thaliana and the blade of arabidopsis VTC1 mutant vtc1-1, rinse well with water, and blot with absorbent paper
Unnecessary moisture, uses liquid nitrogen quick freeze the most respectively, in-80 DEG C of preservations.
Two, accurately weigh 0.175g AsA, be dissolved in the perchloric acid (HClO that 1ml volumn concentration is 6%4) aqueous solution
In, obtain the AsA solution of 1mmol/ml.
Three, it is finally diluted to by the perchloric acid solution that AsA solution volumn concentration is 6% of 1mmol/ml
Concentration be respectively 1000nmol/ml, 800nmol/ml, 600nmol/ml, 400nmol/ml, 200nmol/ml,
The AsA solution of 100nmol/ml.
Four, take the AsA solution of the variable concentrations that 300 μ l step 3 obtain respectively, add 2700 μ l pH=12.7
In sodium succinate buffer, mixing, the concentration of about pH=5.8 now, AsA decreases 10 times accordingly.At 265nm
Place surveys the absorption value of the AsA of variable concentrations.With absorption value (OD) as abscissa, AsA concentration is vertical coordinate mapping,
Draw standard curve, obtain equation of linear regression.
Five, take the material of each plant of the 0.5g that step one obtains respectively, at pulverized under liquid nitrogen, be separately added into 1ml
Volumn concentration is the HClO of 6%4Aqueous solution, mixing, place 5min on ice, 4 DEG C, 12000g, centrifugal 10min.
Take supernatant, extract AsA therein.
Six, the supernatant 200 μ l that step 5 is extracted is added in 1800 μ l pH=12.7 sodium succinate buffer, so
The ascorbic acid oxidase (purchased from Sigma) of rear addition 4U, after reaction 10min, records 265nm ultraviolet absorption value,
It is denoted as OD1；The supernatant 200 μ l taking step 5 extraction adds in 1800 μ l pH=12.7 sodium succinate buffer,
Add DTT extremely final concentration of 30mM, room temperature reaction 30min, record 265nm ultraviolet absorption value, be denoted as OD2.
Seven, equation of linear regression OD2-OD1 value substitution step 4 obtained, calculates the concentration of AsA.
Vitamin C content testing result in each plant is as shown in Figure 7.
In Fig. 7 A, WT is wildtype Arabidopsis thaliana, and vtc1-1 is the single-point mutants vtc1-1, OE-1 of arabidopsis VTC1
It is the arabidopsis two strain T3 generations turning OsVTC1-3 over-express vector pCAMBIA1307-OsVTC1-3 with OE-3.
In Fig. 7 B, WT be wild rice is spent 17, RI-2 and RI-4 be two strain T3 generation turn OsVTC1-3 interference
The Oryza sativa L. of carrier pCAMBIA2300-RNAi-OsVTC1-3.
Fig. 7 shows, OsVTC1-3 can improve Vc content in arabidopsis VTC1 mutant vtc1-1, shows
OsVTC1-3 albumen has VTC1 similar functions in plant, improves plant Vc by catalysis GDP-mannose synthesis and synthesizes.
The expression inhibiting of OsVTC1-3 reduces the synthesis of Oryza sativa L. Vc, when the expression inhibiting of OsVTC1-3, and Vc in its root
Content is about wild rice 50%, it is seen that OsVTC1-3 has important function in Oryza sativa L. Vc synthesizes.
Claims (1)
1. the method preparing the transgenic plant that ascorbic acid biosynthesis ability reduces, comprises the steps:
Being imported by interference carrier sets out in plant, obtains transgenic plant；Compared with the described plant that sets out, the ascorbic acid biosynthesis ability of described transgenic plant reduces；
The building process of described interference carrier is: is inserted by the DNA fragmentation shown in SEQ ID No.3 and sets out carrier pUCCRNAi'sXhoI HeBglBetween II site, obtain intermediate carrier 1；The reverse complementary sequence of the DNA fragmentation shown in SEQ ID No.3 is inserted described intermediate carrier 1SalI HeBamHBetween I site, obtain intermediate carrier 2；WithPstDescribed in I enzyme action, intermediate carrier 2, obtains DNA fragmentation；PstI enzyme action pCAMBIA2300, obtains carrier large fragment；Described DNA fragmentation is connected with described carrier large fragment, finally gives described interference carrier；
Described plant is Oryza sativa L..
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310699609.8A CN103710380B (en) | 2013-12-18 | 2013-12-18 | The application in promoting plant vitamin C synthesis of the OsVTC1-3 albumen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310699609.8A CN103710380B (en) | 2013-12-18 | 2013-12-18 | The application in promoting plant vitamin C synthesis of the OsVTC1-3 albumen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103710380A CN103710380A (en) | 2014-04-09 |
| CN103710380B true CN103710380B (en) | 2016-08-17 |
Family
ID=50403734
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310699609.8A Expired - Fee Related CN103710380B (en) | 2013-12-18 | 2013-12-18 | The application in promoting plant vitamin C synthesis of the OsVTC1-3 albumen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103710380B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107474124B (en) * | 2017-08-25 | 2020-04-07 | 中国农业科学院生物技术研究所 | Application of OsAPBP2 protein in promoting synthesis of plant folic acid |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792345A (en) * | 2010-03-20 | 2010-08-04 | 邓正春 | Production method of vitamin Zn-Se fertilizer and using method thereof |
| CN103250695A (en) * | 2013-05-24 | 2013-08-21 | 广东省农业科学院水稻研究所 | Natural plant extract rice seed embryo vigor retention agent as well as preparation method and application thereof |
-
2013
- 2013-12-18 CN CN201310699609.8A patent/CN103710380B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792345A (en) * | 2010-03-20 | 2010-08-04 | 邓正春 | Production method of vitamin Zn-Se fertilizer and using method thereof |
| CN103250695A (en) * | 2013-05-24 | 2013-08-21 | 广东省农业科学院水稻研究所 | Natural plant extract rice seed embryo vigor retention agent as well as preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 水稻OsJAB1与OsVTC1的相互作用;张传玉;《中国优秀硕士学位论文全文数据库农业科技辑》;20111215(第12期);摘要，正文"1.4 植物中维生素C生物合成的调控" * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103710380A (en) | 2014-04-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102888425B (en) | Method for producing astaxanthin by using transgenic plant | |
| Zhang et al. | A newly isolated Na+/H+ antiporter gene, DmNHX1, confers salt tolerance when expressed transiently in Nicotiana benthamiana or stably in Arabidopsis thaliana | |
| CN114525284B (en) | Red skin longan anthocyanin biosynthesis regulatory gene DlMYB1-HP and application thereof | |
| CN106399114B (en) | A kind of method and application improving DNC wireless ethanol tolerance ability | |
| Yang et al. | Comparative study of vegetative and reproductive growth of different tea varieties response to different fluoride concentrations stress | |
| CN110643630A (en) | Application of KNAT1 gene in improving salt stress resistance of plants | |
| CN103710380B (en) | The application in promoting plant vitamin C synthesis of the OsVTC1-3 albumen | |
| CN102558325B (en) | The preparation method of sweet wormwood AaORA albumen and encoding gene, transgene abrotanum plant | |
| Vijayraghavan et al. | Effect of short-and long-term phosphate stress on the non-phosphorylating pathway of mitochondrial electron transport in Arabidopsis thaliana | |
| CN108707594B (en) | 1 gene family of -1 related protein kinase of maize sucrose non-fermented and its application | |
| CN106191001A (en) | The application in improving plant salt endurance of phospholipase PLD ζ 1 gene | |
| CN114085854B (en) | Drought-resistant and salt-tolerant gene OsSKL2 for rice and application thereof | |
| CN105950644B (en) | The albumen and application of asparagus enzyme, namely chalcone isomerase gene and its coding | |
| CN113005138A (en) | Arabidopsis PHO1; application of H10 protein and coding gene thereof in regulation and control of plant leaf anthocyanin synthesis | |
| CN103421809A (en) | Application of OsHSF08 gene in controlling rice drought resistance | |
| CN102499037B (en) | Method for rapid propagation of genetically modified sweet wormwood by hydroponics | |
| CN104004078A (en) | Application of OsVTC1-1 in improvement of plant salt stress tolerance | |
| CN110241122A (en) | Application of the Caragana intermedia CiCPK10 gene in regulation stress resistance of plant | |
| CN106831967A (en) | Reduce IAA10 albumen and its encoding gene expression is improving plant to the application in fractilinea oryzae resistance | |
| CN105936914B (en) | The albumen and application of asparagus chalcone synthase genes and its coding | |
| CN102559631B (en) | Malus xiaojinensis Cheng et Jiang MxHA5 protein, and coding gene and application thereof | |
| CN104988175B (en) | Application of the tomato HsfA1a genes in plant autophagosome activity and drought resistance is improved | |
| CN103820475A (en) | Geranylgeranyl pyrophosphate synthetase gene in Lycium chinense Miller, and encoded protein and application thereof | |
| CN106811446A (en) | Paddy rice arsenic acid reductase gene OsHAC4 and its application | |
| CN106834343B (en) | Sucrose synthase is in the regulation developmental application of fruit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160817 Termination date: 20191218 |